Der3p/Hrd1p is required for endoplasmic reticulum-associated degradation of misfolded lumenal and integral membrane proteins

Abstract

We have studied components of the endoplasmic reticulum (ER) proofreading and degradation system in the yeast Saccharomyces cerevisiae. Using a der3-1 mutant defective in the degradation of a mutated lumenal protein, carboxypeptidase yscY (CPY*), a gene was cloned which encodes a 64-kDa protein of the ER membrane. Der3p was found to be identical with Hrd1p, a protein identified to be necessary for degradation of HMG-CoA reductase. Der3p contains five putative transmembrane domains and a long hydrophilic C-terminal tail containing a RING-H2 finger domain which is oriented to the ER lumen. Deletion of DER3 leads to an accumulation of CPY* inside the ER due to a complete block of its degradation. In addition, a DER3 null mutant allele suppresses the temperature-dependent growth phenotype of a mutant carrying the sec61-2 allele. This is accompanied by the stabilization of the Sec61-2 mutant protein. In contrast, overproduction of Der3p is lethal in a sec61-2 strain at the permissive temperature of 25 degrees C. A mutant Der3p lacking 114 amino acids of the lumenal tail including the RING-H2 finger domain is unable to mediate degradation of CPY* and Sec61-2p. We propose that Der3p acts prior to retrograde transport of ER membrane and lumenal proteins to the cytoplasm where they are subject to degradation via the ubiquitin-proteasome system. Interestingly, in ubc6-ubc7 double mutants, CPY* accumulates in the ER, indicating the necessity of an intact cytoplasmic proteolysis machinery for retrograde transport of CPY*. Der3p might serve as a component programming the translocon for retrograde transport of ER proteins, or it might be involved in recognition through its lumenal RING-H2 motif of proteins of the ER that are destined for degradation.

Figure 1

Structure of the DER3 gene. Nucleotide sequence of DER3 and the predicted amino acid sequence of Der3p are shown. The regions with the predicted transmembrane domains are underlined; the H2-RING finger motif is marked with bold letters.

Figure 2

(A) Steady-state levels of CPY* in DER3 wild-type and mutant strains. Western blotting was performed with crude extracts prepared from early stationary phase yeast cells grown on CM medium of the strains YAF29 (der3–1), YAF29 harboring the centromeric complementing plasmid YCpDER3, wild-type strain W303–1C (DER3), and DER3 deleted strain W303–1CΔ3 (Δder3). (B) Kinetics of CPY* degradation in DER3 wild-type and mutant strains. A pulse-chase experiment performed with the wild-type strain W303–1C (DER3) and W303–1CΔ3 (Δder3) is shown. Cells were labeled for 20 min with [³⁵S]methionine, and samples were taken at the indicated chase periods. (C) Steady-state levels of CPY* in DER1 and DER3 mutant and wild-type strains. Crude extracts from early stationary phase cells of the strains W303–1C (wild-type), W303–1CD (Δder1), W303–1CΔ3 (Δder3), and W303–1CDΔ3 (Δder1 Δder3) were prepared. CPY-immunoreactive material was detected after SDS-PAGE and Western blotting with monoclonal anti-CPY antibodies.

Figure 3

(A) Identification of the Der3 protein. Crude extracts of exponentially growing cells on CM medium of strains W303–1C [wild type (WT)], W3031-C harboring the high-copy plasmid YEpDER3, YAF29 (der3–1), W303–1CΔ3 (Δder3), and W303–1CD (Δder1) were prepared, separated by SDS-PAGE, and immunoblotted. Levels of Der3p were detected with polyclonal Der3p antisera. (B) Der3p is an integral membrane protein. Crude extracts enriched in membranes were prepared from exponentially growing cells on CM medium of strains W303–1C (WT) and W303–1CΔ3 (Δder3). Aliquots of the soluble (S) and membrane (P) fractions were loaded onto a 8% SDS-polyacrylamide gel and separated by electrophoresis followed by Western blotting. Der3p antigenic material was visualized with anti-Der3p polyclonal antibodies.

Figure 4

Intracellular localization of Der3p. (A) Der3p cofractionates with the ER resident protein Kar2p. Spheroplasts of the W303–1C (wild-type) strain harboring the high-copy plasmid YEpDER3 were prepared, and, after a gentle lysis, the homogenate was fractionated on a 10-step sucrose gradient (18–54%, steps of 4% sucrose). Aliquots of each fraction were subjected to SDS-PAGE and Western blotting. Levels of Der3p, Kar2p (ER marker), and the 100-kDa subunit of the vacuolar membrane ATPase were detected with the respective specific antibodies. The activity of GDPase, a Golgi marker, is given as percentage of the highest activity value measured. (B) Der3p is localized in ER-like structures. Exponentially growing cells of the strain W303–1C transformed with the 2 μ plasmid YEpDER3 were fixed and stained with polyclonal anti-Der3p antibodies and goat anti-rabbit Cy3 antibody. Cy3 fluorescence was monitored with a laser confocal microscope (Cy3, Cy3 fluorescence; DIC, Nomarski optics). (C) Also when expressed from the chromosomal copy of the gene, Der3p cofractionates with Kar2p. A subcellular fractionation was performed as in A using strain W303–1C. For detection of Der3p, 500 μl of each fraction were diluted up to 10-fold with 10 mM HEPES (pH 7.5) and the 100,000-g membrane pellets (1 h) were resuspended in urea buffer and loaded onto a 8% SDS-polyacrylamide gel. After Western blotting Der3p and Kar2p were detected with suitable antibodies. Only fractions 1–8 are shown; no band could be detected in fractions 9–17.

Figure 5

The C-terminal tail of Der3p is oriented to the ER lumen. Yeast spheroplasts of the wild-type strain W303–1C were subjected to gentle lysis. The lysate was centrifuged to separate a soluble fraction (S) from the pellet fraction (P) containing intact yeast microsomes. Aliquots of microsomes were incubated for 30 min on ice in the absence (−) or in the presence of trypsin (+T) or trypsin and Triton X-100 (+T+X). Treatments were stopped by trichloroacetic acid precipitation (10% final concentration). Pellets were resuspended in urea buffer, immunoblotted after SDS-PAGE, and stained with anti-Der3p or anti-Kar2p polyclonal antibodies.

Figure 6

Localization of CPY* in different mutants with a reduced ER degradation. Protease protection experiments were performed with intact yeast microsomes of the strains W303–1CΔ3 (Δder3), W303–1CD (Δder1), W303-CQ (Δubc7), and W303CPQ (Δubc6 Δubc7). Aliquots of the soluble fraction (S) and ER vesicles (P) not treated (−) or treated with trypsin (+T) or with trypsin and Triton X-100 (+T+X) were subjected to Western blotting and levels of CPY* and for control Kar2p were detected with suitable antibodies.

Figure 7

The Ubc6-Ubc7 ubiquitination and proteasome pathway is not affected by the absence of Der3p. The kinetics of the degradation of a Deg1-β-galactosidase fusion protein in the strains W303–1C (WT) and W303–1CΔ3 (Δder3) was measured. Early stationary phase yeast cells grown on CM medium of both strains harboring the high-copy plasmid pDeg1 were harvested and resuspended in fresh CM medium containing cycloheximide (0.5 mg/ml) and incubated at 30°C. Samples of the culture were taken after 0, 30, 60, and 90 min, and the activity of β-galactosidase was measured. Enzymatic activity was set at 100% at time 0.

Figure 8

(A) Disruption of DER3 suppresses the temperature-sensitive growth phenotype of the sec61–2 mutant. Strains W303–1C (WT), YRP086 (sec61–2) and YRP105 (sec61–2 Δder3) harboring the LEU2 containing plasmid YCplac111, and YRP105 (sec61–2 Δder3/YCpDER3) containing the DER3 gene in a centromeric plasmid were tested for growth at 30 and 38°C on CM plates without leucine for 36 h. (B) Deletion of DER3 blocks the degradation of Sec61–2p at restrictive temperature. Pulse-chase experiments were performed in strains W303-1C (WT), YRP086 (sec61–2), and YRP105 (sec61–2 Δder3). Exponentially growing cells on SD medium were shifted to 38°C for 2 h and labeled with [³⁵S]methionine. After the chase aliquots of cells were taken at the indicated chase points, lysed, and immunoprecipitated with specific anti-Sec61p antibodies.

Figure 9

Overexpression of DER3 is lethal in a sec61–2 mutant at permissive temperature. Strains W303–1C (WT) and YRP086 (sec61–2) harboring the URA3 containing plasmid pBM150 or the DER3 gene under the control of the GAL1 promoter cloned into pBM150 plasmid (pBM/DER3) were grown on CM plates without uracil with glucose or galactose as carbon source at 25 and 30°C for 36 h.

Figure 10

Integrity of the lumenal tail of Der3p is required for a proper degradation of CPY* and Sec61–2p. (A) The C-terminal hydrophilic tail of the truncated protein Der3ΔR is oriented to the ER lumen. A protease protection assay was performed with intact yeast microsomes from strain W303–1CΔ3 harboring the plasmid YCpDER3ΔR. Levels of Der3ΔRp and Kar2p were detected after Western blotting in the soluble fraction (S) and in ER vesicles (P) not treated (−) or after treatment with trypsin (+T) or with trypsin and Triton X-100 (+T+X). (B) Steady-state levels of CPY* in crude extracts prepared from yeast cells from strain W303–1C expressing wild-type Der3p (DER3), W303–1CΔ3 (Δder3), and W303–1CΔ3 harboring the plasmid YCpDER3ΔR (DER3ΔR) grown on CM medium were immunodetected with monoclonal anti-CPY antibodies after SDS-PAGE using Western blotting. (C) Expression of the truncated Der3ΔR protein is not able to revert the suppression of the temperature-sensitive growth phenotype caused by the disruption of the DER3 gene. Strains W303–1C (WT) and YRP086 (sec61–2) harboring the LEU2-based centromeric plasmid YCplac111 and strain YRP105 containing the plasmids YCplac111(sec61–2Δder3), YCpDER3 (sec61–2Δder3/YCpDER3), or YCpDER3ΔR (sec61–2Δder3/YCpDER3ΔR) were tested for growth on CM plates without leucine at 30and 38°C for 36 h. (D) Der3ΔR protein cannot support degradation of the mutated Sec61–2p at 38°C. Exponentially growing cells on CM medium at 25°C from strains W303–1C (WT/DER3), YRP086 (sec61–2/DER3), YRP105 (sec61–2/Δder3), and YRP105 harboring the plasmid YCpDER3ΔR (sec61–2/DER3ΔR) were shifted to 38°C for 1 h. Levels of Sec61p were detected after SDS-PAGE and Western blotting with specific antibodies.